Agglutination tests can be used as qualitative tests to assay for the presence of an antigen or an antibody or as quantitative tests to measure the level of antibodies to particulate antigens. Rapid Plate Tests (RPT) or Plate Agglutination Tests (PAT)/Slide Agglutination Tests (SAT) are screening tests used to detect antibodies to microorganisms in the sera. Positive serum samples are subjected to Tube Agglutination Test (TAT) for further confirmation and quantitation of the titer of antibodies. In quantitative test, serial dilutions are made of a serum sample to be tested for antibody and then a fixed amount of particulate antigen is added. The maximum dilution that gives visible agglutination is called the titer. The intensity of the agglutination reaction is a good indicator of the concentration of antibody in the serum. Very low concentration of antibodies may not give visible agglutination. The lack of agglutination at high concentrations of antigen or antibodies is called the Prozone effect. In Prozone very small complexes are formed that do not clump to form visible agglutination. These factors also lead to false negative results.
In many countries, the standard Plate Agglutination Test is the routine test for human Brucellosis. However, it may give false negative results (WHO Report, 1986; Lucero and Bolpe, 1998). Rose Bengal Plate Test (RBPT) is a variant of plate/slide agglutination test where killed Brucella organisms stained with Rose Bengal dye are used as antigen for detection of antibodies in the serum. The International Office of Epizootics has recommended the RBPT as one of the tests for the diagnosis of bovine Brucellosis (Corbel and MacMillan, 1995). The RBPT is a quick, cheap and effective test for the diagnosis of Brucellosis. It can be carried out with the minimum of equipment, and the end result is read by the naked eye. However, many factors affect the RBPT reactions and their reading. Some people are able to see the finer agglutination while many others cannot. This causes variation in results. Some authors (Saravi et al., 1990) have reported unacceptable rate of false negatives with the RBPT. The sensitivity of RBPT antigens obtained from different sources may vary considerably when used for testing sera from animals in herds/flocks of low prevalence (Blasco et al., 1994).
The conventional agglutination test is a simple and cheap method for diagnosis of infectious diseases. However, it has lesser sensitivity and hence more chances of false negative results compared to other serological tests like ELISA, Western Blotting etc. Earlier attempts to enhance agglutination have led to the development of Indirect Coombs' Test and Coagglutination Test. The Indirect Coombs' Test using antiglobulin has been applied for cross linking of serum antibodies to make larger clumps of agglutinates for easy detection. However, antiglobulin alone forms spread-out meshwork of antibodies with loose ends of antiglobulin which are difficult to detect with the naked eye. Coagglutination is a sensitive test for screening and rapid diagnosis of infectious diseases. However, it requires Protein A which binds to the immunoglobulins in a non-specific manner and is expensive as well as cumbersome to prepare. Furthermore, false positive results are possible in both, the Coombs' ntiglobulin Test as well as the Coagglutination Test due to the inability to differentiate non-specific aggregates of antigen particles alone from the specific agglutinates formed by antigen and antibody combined.
Limitations of the currently available diagnostic tests include:                a) The available agglutination tests and kits based on them have less sensitivity and more chances of false negative results.        b) Inability to differentiate non-specific aggregates of particulate antigen formed by improper mixing from the specific agglutinates of antigen and antibody may lead to false positive results in conventional agglutination tests.        c) The Indirect Coombs' test using antiglobulin has been applied for cross linking of serum antibodies to make larger clumps of agglutinates for easy detection. However, antiglobulin alone forms widely spread-out meshwork of antigen-antibody clumps with loose ends of antibody bound antiglobulin difficult to detect with the naked eye.        d) Coagglutination Test is dependent on Protein A which is expensive and binds non-specifically to immunoglobulins.        e) False positive results are possible in both, the Coombs' Antiglobulin Test as well as the Coagglutination Test due to the inability to differentiate non-specific aggregates of antigen particles alone from the specific agglutinates formed by antigen and antibody combined.        f) ELISA and Western Blotting require trained technicians and well equipped labs which are few in developing countries. Kits based on these assays are expensive.        g) Molecular diagnostic techniques can be performed only by well trained technicians in well equipped laboratories which are few in developing countries. Commercially available kits based on these assays are expensive.        